The invention concerns an arrangement and a method of examining hydrophilic macromolecules in an aqueous solution having a solid carrier surface provided with a lipid film, wherein the molecules to be examined are bound to the lipid film by means of a molecular coupling system and thus are rendered immobile.
Such an arrangement and a corresponding operational method are known from Liu et al., Eur. Biophys. (J. 1995) 24: 31-38.
Hydrophilic proteins have been investigated for some time by means of FT-IR-ATR technology. In this connection, they are usually disposed in dissolved form onto an ATR crystal and are immobilized by drying. Then, FT-IR spectra are recorded from the dried protein films. The dried proteins are dissolved by adding water. In order to be able to study the interaction between hydrophilic proteins and other molecules, the proteins have to be present in the aqueous environment in their native form. As a consequence, for such investigations by means of ATR technology, the proteins should be present in a form bound to the ATR crystal in a "water-proof" manner.
From Blankenburg et al., Biochemistry (1989) 28: 8214-8221 an arrangement is known for the examination of streptavidin by means of UV light in an area of 230 nm, wherein the extremely high affinity of the protein streptavidin for the cyclic urea derivative biotin (vitamin H; bonding constant: 10.sup.15 M.sup.-1), which has been known for more than 20 years and has been utilized in connection with the most different applications in biotechnology, is utilized for attaching streptavidin to be examined to biotinylated phospholipids. In this known method, the lipids are arranged again in a monolayer on a water surface.
Darst et al., Biophys. J. (1991) 59: 387-396 disclose on the one hand an arrangement for the examination of lipid films by means of electron diffraction, wherein crystalline streptavidin is arranged on a teflon trough and, on its side facing away from the teflon trough, is bound to biotinylated lipids. Furthermore, this publications also discloses an arrangement in which the hydrophilic protein Ferritin is bound to crystallized streptavidin by biotin residues. In this connection, the use of lipids in the arrangement to be examined is again not mentioned.
The publication "PE Applied Biosystems report" of the company Perkin Elmer, 1/1997, page 21, describes an arrangement for the examination of interactions between membranes and proteins in an aqueous medium, in which by means of weakened total reflection infrared spectroscopy (ATR-IR) the electrostatic connection of the protein Trypsin Inhibitor to a membrane in buffered water with defined pH value was measured. To this end, a lipid double layer of a thickness of approximately 5 nm (so-called solid-supported membrane=simple model of a biological membrane) was applied to the surface of a Germanium-ATR-crystal, and on the surface thereof facing away from the ATR crystal, the protein to be examined, from an aqueous solution, was added.
The initially cited article by Liu et al., Eur. Biophys. J. (1995) 24, 31-38 finally describes a system, wherein a lipid monolayer is arranged on a hydrophobic silicon surface, which is biotinylated on its side facing away from the silicon, wherein the biotin molecules again are bound to avidin molecules. The latter are examined optically by means of dispersion of He--Ne laser light in an area of 633 nm. This system is suited only for measurements with one particular protein, namely avidin, which can be immobilized on the biotin lipid film due to its high bonding affinity for biotin.
In contrast thereto, it is the object of the present invention to present an arrangement and a method of the initially mentioned type, which enable immobilization and examination of any hydrophilic macromolecules, wherein it should be possible to coat the solid carrier surface in an as easy as possible and reversible manner.